ANTIMICROBIALAGENTS ANDCHEMOTHERAPY, May 2009, p. 2149–2152 Vol. 53, No. 5 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00693-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Violacein Extracted from
Chromobacterium violaceum
Inhibits
Plasmodium
Growth In Vitro and In Vivo
䌤
Stefanie C. P. Lopes,
1,2Yara C. Blanco,
1,2Giselle Z. Justo,
3Paulo A. Nogueira,
4†
Francisco L. S. Rodrigues,
4Uta Goelnitz,
5Gerhard Wunderlich,
5Gustavo Facchini,
6Marcelo Brocchi,
1Nelson Duran,
7and Fabio T. M. Costa
1,2*
Departamento de Microbiologia e Imunologia, Instituto de Biologia, IB, Universidade Estadual de Campinas, UNICAMP, P.O. Box 6109, 13083-970 Campinas, SP, Brazil1; Departamento de Parasitologia, IB, UNICAMP, P.O. Box 6109, 13083-970 Campinas, SP, Brazil2;
Departamento de Bioquímica, Universidade Federal de Sa˜o Paulo, UNIFESP, Rua 3 de Maio 100, 04044-020 Sa˜o Paulo, SP, Brazil3; IPEPATRO/CEPEM, BR 364, KM 4.5, 78900-970 Porto Velho, RO, Brazil4; Departamento de Parasitologia, ICB-2,
Universidade de Sa˜o Paulo, USP, Sa˜o Paulo, SP, Brazil5; Departamento de Fisiologia e Biofísica, IB, UNICAMP, P.O. Box 6109, 13083-970 Campinas, SP, Brazil6; and Instituto de Química, Laborato´rio de
Química Biolo´gica, IQ, UNICAMP, C.P. 6154, 13083-970 Campinas, SP, Brazil7
Received 27 May 2008/Returned for modification 30 August 2008/Accepted 24 February 2009
Violacein is a violet pigment extracted from the gram-negative bacteriumChromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistantPlasmodium falciparumstrains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protectedPlasmodium chabaudi chabaudi -infected mice from a lethal challenge.
Violacein is a violet pigment isolated fromChromobacterium violaceum, a gram-negative betaproteobacterium found in the Amazon River in Brazil. It has been reported to kill bacteria (4) and induces apoptosis in various types of cancer cells (1, 5, 7, 8, 10, 11). Moderate activity againstTrypanosoma cruziand
Leishmania amazonensispromastigotes has also been observed (3, 9). Due to the widespread presence of drug resistance in the malaria parasite, resulting in dramatically decreased efficacy of available antimalarial drugs (15), and the fact that immuno-protection achieved by the most successful malaria vaccine is only partial and short-lived (14), we evaluated the in vitro and in vivo effects of violacein on human and murine blood stage forms ofPlasmodiumparasites.
Isolation and purification of violacein, 3-[1,2-dihydro-5-(5-hy-droxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydeno]-1,3-dihydro-2H -indol-2-one (Fig. 1), fromC.violaceum (CCT3496) were per-formed as previously described (12). Toxicity was measured as the concentration-dependent lysis of normal erythrocytes (NE) by counting red blood cells per milliliter with the aid of a Neubauer chamber. After 48 h of exposure to various concen-trations of violacein, the percent red blood cell density (RBCD) relative to that of the control (without violacein) was monitored and calculated according to the formula (RBCD per milliliter in the presence of violacein/RBCD per milliliter without violacein)⫻100. As shown in Fig. 2A, a slight reduc-tion in the RBCD percentage at violacein concentrareduc-tions of
⬎8.0M was observed. Significant (Mann-Whitney U test,P⬍
0.05) toxicity to NE occurred at a concentration of 14.0M. Next, we performed dose-response assays to obtain the 50% inhibitory concentrations (IC50s) of violacein against
erythro-cytes infected with chloroquine-sensitive or -resistant strains of
P.falciparum (3D7 [16] or S20 [2], respectively) at 1% para-sitemia and a 2% final hematocrit. We used [3
H]hypoxanthine (Amersham Biosciences, Amersham, United Kingdom) incor-poration to assess parasite growth according to a protocol described elsewhere (13). Violacein was tested in triplicate at least three times with different batches and cells, and parasite growth was compared to that in nontreated infected erythro-cytes (IE), which represented 100% parasite growth. Percent parasite growth inhibition was calculated according to the for-mula [1⫺(cpm of treated IE⫺cpm of NE/cpm of nontreated IE⫺ cpm of NE)]⫻100. After a 48-h incubation, violacein inhibited parasite development even at the lowest tested con-centration of 0.06M and completely abrogated parasite via-bility at concentrations of⬎1.0M (Fig. 2B).
The IC50of violacein againstP.falciparum strain 3D7 was
calculated as 0.85 ⫾ 0.11 M. We then tested whether the effect of violacein was directed against young (rings, 0 to 24 h) or mature (trophozoites and schizonts, 24 to 48 h) blood forms by using synchronized parasites (⫾6 h) obtained by repeated 5% sorbitol treatment as previously described (6). After a 24-h incubation, inhibition of the different parasite stages by viola-cein was measured. As shown in Fig. 2C, there was no statis-tically significant difference (Mann-Whitney U test,P⬎0.05) between the inhibitory values of violacein in either parasite stage. We then asked if susceptibility or resistance to chloro-quine also predicts parasite sensitivity to violacein. As shown in Fig. 2D, the violacein IC50s for strains 3D7 and S20 did not
differ significantly (0.85⫾0.11 and 0.63⫾0.13M, respec-tively; Mann-Whitney U test,P⬎0.05).
* Corresponding author. Mailing address: Departamento de Parasi-tologia, Instituto de Biologia, IB, Universidade Estadual de Campinas, UNICAMP, P.O. Box 6109, 13083-970 Campinas, SP, Brazil. Phone: 55 19 3788-6594. Fax: 55 19 3788-6276. E-mail: costaftm@unicamp.br. † Present address: Centro de Pesquisa Leonidas & Maria Deane, FIOCRUZ, Laborato´rio de Biodiversidade em Sau´de, 69057-070 Manaus, AM, Brazil.
䌤Published ahead of print on 9 March 2009.
We then investigated whether violacein antimalarial activity could be sustained in a mouse model, where other character-istics such as bioavailability and pharmacokinetics have to be taken in account. For the in vivo assays, C57BL/6 mice (7 to 10 mice per group, aged 7 to 10 weeks, and with a body weight of 20⫾3 g) were infected with a nonlethal (AS) or a lethal (AJ) strain of Plasmodium chabaudi chabaudi by intraperitoneal (i.p.) injection with 106
IE. Parasitemia levels were determined daily by counting the IE among at least 1,000 erythrocytes in Giemsa-stained blood smears. As shown in Fig. 3A, daily
ad-ministration of violacein i.p. for 11 consecutive days (0 to 10 days postinfection [p.i.]) reduced the parasitemia of P.
chabaudi chabaudiAS-infected mice. Thirty-nine percent in-hibition was observed on day 7 p.i. (parasitemia peak), in comparison to nontreated mice (control; Table 1), even at a low dose of 0.75 mg/kg/day. Moreover, the two highest doses of violacein (3.75 and 7.5 mg/kg/day) almost completely abolished parasitemia on day 7 p.i., corresponding, respectively, to 82 and 87% inhibition of parasite development (Table 1). In ad-dition, violacein doses of 0.75 to 7.5 mg/kg/day were able to inhibit the peak parasitemia in a dose-dependent manner (Ta-ble 1).
Since violacein did not completely abrogate parasitemia early in infection and drug pressure was removed by day 10 p.i., we monitored the parasitemia levels ofP.chabaudi chabaudi
AS-infected mice treated with the highest dose of violacein daily until day 22 p.i. Notably, on day 16 p.i., which represents the sixth day after the end of violacein treatment, parasite development was still significantly (Mann-Whitney U test,P⬍ FIG. 1. Chemical structure of violacein.
FIG. 2. Evaluation of violacein antimalarial activity againstP.falciparum. (A) Noninfected erythrocytes (RBC) were cultivated for 48 h at 37°C in the presence of different concentrations of violacein. The RBCD was determined as a percentage of that the control (without drug). Inhibition of the growth ofP.falciparum3D7 IE cultivated for 48 h at 37°C with different concentrations of violacein (B) and of young or mature stages of this parasite (C) is also shown. (D) Effect of violacein against a chloroquine-resistant strain (S20) ofP.falciparumin IE. Results are expressed as the mean of triplicate measurements⫾the standard deviation.
0.05) inhibited (up to 59%; Fig. 3B). To verify whether viola-cein had an effect on parasite growth after the establishment of infection,P.chabaudi chabaudiAS-infected mice received vio-lacein from day 5 (16% parasitemia) to day 10 p.i. This can
reflect the time point when malaria therapy is given to patients. As shown in Fig. 3C, violacein administration during patent parasitemia was able to reduce parasite growth significantly (Mann-Whitney U test,P⬍0.01), by up to 50.1%, in
compar-FIG. 3. Effect of violacein on mouse-derivedPlasmodiumparasites. Groups of 7 to 10 C57BL/6 mice were infected i.p. with 106P.chabaudi chabaudiAS IE and then left untreated or treated with different doses of violacein administered i.p. for 11 consecutive days (days 0 to 10 p.i.), starting on day 0 at 1 h p.i. Parasitemia levels were determined daily until day 12 p.i. (A) or up to day 22 p.i. (B) after treatment with the highest dose (7.5 mg/kg/day). (C) Analysis of violacein antimalarial activity after parasite establishment and treatment for 6 consecutive days (days 5 to 10 p.i.) with the highest dose of violacein inP.chabaudi chabaudiAS-infected mice. Results are expressed as the mean of a group of mice⫾the standard deviation. (D) Analysis of the survival of groups of 10 C57BL/6 mice infected withP.chabaudi chabaudiAJ (lethal strain) by i.p. injection of 106IE and then treated i.p. with violacein at 7.5 mg/kg/day for 11 consecutive days (days 0 to 10 p.i.). The drug administration period is indicated
by shading in each graph.
TABLE 1. Inhibition of parasitemia inP.chabaudi chabaudiAS-infected mice under violacein treatment
Violacein dose (mg/kg/day)
Mean % inhibitiona⫾SD (
Pvalue)
Day 5 Day 6 Day 7 Day 8
7.5 56.48⫾12.84 (⬍0.005) 75.40⫾5.17 (⬍0.005) 86.90⫾3.53 (⬍0.005) 84.67⫾5.43 (⬍0.05) 3.75 33.23⫾14.72 (NS)b 64.89⫾8.00 (⬍0.005) 82.12⫾4.33 (⬍0.005) 75.73⫾13.93 (⬍0.05)
0.75 3.58⫾33.80 (NS) 20.42⫾36.30 (NS) 39.25⫾14.15 (⬍0.05) 48.85⫾15.08 (NS)
0.075 NIc(ND)d NI (ND) NI (ND) 12.04⫾6.11 (NS)
aThe data shown are percentages of the parasitemia of nontreated mice, which was defined as 100%. Values for groups of 7 to 10 mice are shown. bNS, not statistically significant.
cNI, no inhibition. dND, not determined.
ison to nontreated animals on the seventh day of infection. Next, to determine the protective effect of violacein, mice were infected with the lethal AJ strain ofP.chabaudi chabaudiand their survival rate was evaluated. As shown in Fig. 3D, 100% of the nontreated mice died by day 10 p.i., with 50% of the deaths occurring early on day 7 p.i. In contrast, animals treated with violacein at 7.5 mg/kg/day did not succumb to infection until days 9 (10%) and 14 (10%) p.i., reaching 80% survival on day 16; clearly demonstrating the significant (log rank test, P ⬍
0.0001) protective effect of violacein.
This study demonstrates for the first time the antimalarial activity of violacein by showing inhibition of the growth of human- and mouse-derivedPlasmodiumparasites. Also, viola-cein was effective against young and mature forms of the hu-man parasite and its activity extended to chloroquine-sensitive and -resistant strains ofP.falciparum. Our data call for new formulations based on violacein nanoparticles to improve solubility, bioavailability, and activity and to decrease drug toxicity.
This work was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP; grant 2004/00638-6) and the Conselho Nacional de Desenvolvimento Científico e Tecnolo´gico (CNPq; grant 470587/2006-7). S.C.P.L. and Y.C.B. were supported by the Coorde-nac¸a˜o de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), and U.G. and G.F. were sponsored by FAPESP and CNPq fellowships, respectively. F.T.M.C., M.B., and G.W. are CNPq fellows.
We thank Hernando Del Portillo (Department of Parasitology, ICB, USP, Sa˜o Paulo, SP, Brazil) and Maria Regina D’Impe´rio Lima (De-partment of Immunology, ICB, USP, Sa˜o Paulo, SP, Brazil) for kindly providingP.chabaudi chabaudistrains AS and AJ, respectively, and Lindsay Ann Pirrit for revising the English. Many thanks to Carmen L. Lopes and Zeci S. Costa (in memoriam).
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